T cell differentiation in the thymus involves a complex process of positive and negative selection whereby small numbers of functionally- mature T cells are drawn from a large pool of immature precursor cells. Although thymic selection has been a topic of intensive investigation in recent years, many questions remained to be resolved. The following three questions will be addressed in this proposal. What is the mechanism of positive selection of thymocytes? Most workers accept that positive selection occurs in the thymic cortex and reflects T cell contact with "self" major histocompatibility complex (MHC) molecules expressed on cortical epithelial cells. The mechanism of positive selection, however, is poorly understood. To shed light on this issue, T cell differentiation will be studied in a system in which immature thymocytes from normal or T cell receptor (TCR) transgenic mice are cultured in vitro with dissociated populations of purified cortical or medullary epithelial cells. Positive selection will also be studied with a cloned line of thymoma cells. What is the contribution of thymic epithelial cells to negative selection? Although negative selection (tolerance induction) in the thymus is controlled principally by marrow-derived cells, increasing evidence suggests that thymic epithelial cells (TEC) can contribute to tolerance induction. Studies on T cell differentiation in bone-marrow chimeras suggest that selective contact with MHC antigens expressed on TEC induces two forms of tolerance. Some (high-affinity) T cells are deleted in the thymus but other (low-affinity) T cells are rendered anergic. These latter T cells undergo full tolerance induction when the cells encounter host MHC antigens in the post-thymic environment. Various approaches, including studies with thymus-grafted mice and chimeras reconstituted with TCR-transgenic stem cells, will be used to assess the validity of this model. What is the relationship between mature post-thymic T cells and the T cells of the thymic medulla? The relative maturity of T cells in the thymic medulla will be assessed by comparing purified populations of medullary T cells and post-thymic T cells for expression of various surface markers and tolerance susceptibility. The contribution of circulating T cells to medullary T cells will be assessed and attempts will be made to measure T cell export from the thymus in unmanipulated mice. T cells are critical in combating infectious diseases and cancer and it is essential to have precise information on how T cells are formed in the thymus and in what numbers.